There is a growing need for analysis of biomolecules, including proteins, polypeptides and DNA. Capillary electrophoresis (CE) is a process for separating molecules based on their size or charge. In capillary electrophoresis molecules are introduced into a fluid-filled capillary tube and subjected to an electric field (see, Kemp, G. (1998) “CAPILLARY ELECTROPHORESIS: A VERSATILE FAMILY OF ANALYTICAL TECHNIQUES,” Biotechnol. Appl. Biochem. 27:9-17; Wu, D. et al. (1992). Capillary electrophoresis techniques are reviewed by Schwartz, H. et al. (“Separation of Proteins and Peptides by Capillary Electrophoresis: Application to Analytical Biotechnology,” http://www.beckman.com/Literature/BioResearch/727484.pdf),
Capillary electrophoresis (CE) has become an attractive alternative to traditional slab gel electrophoresis for biomolecular separations due to its fast speed and high efficiency. Two primary separation mechanisms are commonly used in CE, separations based on differences in the effective charge of analytes, and separations based on their molecular size. The first separation mechanism is limited to small molecules. Many biomolecules, such as large proteins and DNA are generally separated by molecular sieving electrophoresis and such separations are typically carried out using gel matrices of controlled pore sizes. This technique is also referred as “capillary gel electrophoresis” (“CGE”). The separation achieved using CGE results from the differences in the abilities of different sized molecule to penetrate the gel matrix. Since small molecules move faster than large molecules through the separation gel, size separation is achieved. As for polypeptides and proteins, it is necessary to denature the material (for example, with a sodium dodecyl sulfate (SDS) buffer solution), so that all the proteins will have the same effective charges.
As molecules increase in size the relative differences in their charge diminish. Thus, for larger molecules, such as proteins or nucleic acid molecules, CE is implemented in a manner that accomplishes separation based on size rather than molecular charge. Such size separation is generally accomplished using molecular sieving electrophoresis in which the molecules are drawn through a gel matrix of controlled pore size. It is generally necessary to denature proteins and polypeptides with detergents (e.g., sodium dodecyl sulfate (SDS)), so that disparities in effective charge will not distort the rate with which molecules migrate through the matrix. The process is known as “capillary gel electrophoresis” (“CGE”).
Numerous approaches for accomplishing capillary electrophoresis have been previously described (see, for example, U.S. Pat. Nos.: RE37,606; 6,440,284; 6,436,646; 6,410,668; 6,372,353; 6,358,385; 6,355,709; 6,316,201; 6,306,273; 6,274,089; 6,235,175; 6,153,073; 6,129,826; 6,107,044; 6,074,542; 6,068,752; 6,042,710; 6,033,546; 6,001,232; 5,989,399; 5,976,336; 5,964,995; 5,958,694; 5,948,227; 5,916,426; 5,891,313; 5,846,395; 5,840,388; 5,777,096; 5,741,411; 5,728,282; 5,695,626; 5,665,216; 5,582,705; 5,580,016; 5,567,292; 5,552,028; 5,545,302; 5,534,123; 5,514,543; 5,503,722; 5,423,966; 5,421,980; 5,384,024; 5,374,527; 5,370,777; 5,364,520; 5,332,481; 5,310,462; 5,292,416; 5,292,372; 5,264,101; 5,259,939; 5,139,630; 5,120,413; 5,112,460; 5,015,350; 4,865,706).
U.S. Pat. No. 5,089,111 (Zhu et al.) and U.S. Pat. No. 5,545,302 (Zhu et al.), for example, concern gel-free approaches to capillary electrophoresis. U.S. Pat. No. 5,089,111 (Zhu et al.) discloses an electrophoretic method of separating a mixture of sample ions of varying molecular weights in a sample into components in which the sample is passed through a separation column containing a gel-free aqueous solution of a water-soluble polymer selected from the group consisting of cellulose derivatives, saccharide-based and substituted saccharide-based polymers, polysilanes, polyvinylalcohol and polyvinylpyrrolidone, and in which the polymer has a molecular weight of about 10,000 to about 2,000,000, and is within a range of about 0.1 to about 200 times the average molecular weight of the sample ions in the mixture. The concentration of the polymer in adjusted so as to be sufficient to retard the flow of the species through the separation column to degrees which vary with their molecular weights. The a gel-free aqueous polymer method of U.S. Pat. No. 5,089,111 (Zhu et al.) thus determines the molecular weight of the analytes being separated by retarding the flow of analytes in proportion to their molecular weights. U.S. Pat. No. 5,545,302 (Zhu et al.) concerns a gel-free composition that employs amines to derivative a hydrophilic polymer as a means for reducing endoosmotic flow. The disclosed method concerns suppressing electroendosmotic flow in an electrophoretic separation of a mixture of sample ions in a separation medium consisting essentially of a gel-free aqueous solution, the method comprising including in the gel-free aqueous solution a hydrophilic polymer derivatized by the bonding thereto of an amine at about 0.05 or more equivalents of amine per 100 grams of the polymer. The patent discloses mixing derivatized chains and non-derivatized chains are together.
U.S. Pat. No. 5,264,101 (Demorest et al.) discloses a method of separating biomolecules in a sample comprising preparing a capillary tube with two ends, where the capillary tube (i) has charged chemical groups on its inner wall surface, and (ii) is filled with an electrolyte solution containing 0.05 to 30% weight to weight (w/w) of a non-cross-linked, hydrophilic polymer or copolymer solution containing at least one polymer or copolymer species having (a) a molecular weight between 20 and 5,000 kilodaltons, and (b) a percent charge of between 0.01 to 1.0% as measured by the molar percent of charged monomer subunits to the total polymer subunits, where the charged monomer subunits have the charge opposite to the wall charge at a selected electrophoresis pH, immersing the ends of the tube in anodic and cathodic reservoirs containing an electrolyte solution, introducing a sample containing the biomolecules to be separated into one end of the tube, and applying an electric field across the reservoirs with a polarity effective to fractionate the biomolecules in the sample. The polymers taught by U.S. Pat. No. 5,264,101 (Demorest et al.) are highly ionizable (i.e., they must exhibit a percent charge of between 0.01 to 1.0%) polymers such as amino-acrylamides.
One difficulty encountered in the art is undesirable electroendoosmotic flow and analyte-wall interactions. U.S. Pat. No. 5,567,292 (Madabhushi et al.) discloses a method of suppressing electroendoosmotic flow and analyte-wall interactions to facilitate capillary electrophoresis through the use of water-soluble silica-adsorbing polymers. The disclosed method comprises providing a separation medium containing one or more uncharged water-soluble silica-adsorbing polymers having (i) water solubility in a temperature range between about 20° C. and about 50° C., (ii) a concentration in the separation medium in a range between about 0.001% and about 10% weight/volume, (iii) a molecular weight in the range between about 5×103 and about 1×106 6 daltons, (iv) an absence of charged groups in an aqueous medium having a pH in the range between about 6 and about 9; and employing a separation medium having a viscosity of less than about 1000 centipoise. U.S. Pat. No. 6,358,385 (Madabhushi et al.) concerns a capillary electrophoresis element comprising: a capillary containing an electrophoretic separation medium including a surface interaction component comprising a solution of one or more uncharged water-soluble silica-adsorbing polymers; wherein the inside surface of the capillary is uncoated, and wherein the capillary does not contain a crosslinked polymer gel.
One approach to performing capillary electrophoresis employs charged polymers. U.S. Pat. No. 5,948,227 (Dubrow) concerns a method of separating macromolecules by capillary electrophoresis, comprising: providing a substrate comprising at least a first capillary channel disposed therein, a surface of the channel having a first surface charge associated therewith; filling the capillary channel with a water soluble hydrophilic polymer solution having a percent charge of from about 0.01% to about 2%, as calculated by the molar percent of charged monomer subunits to total monomer utilized in producing the polymer, the charged monomer subunits consist of monomer subunits having a charge that is the same as the first surface charge; introducing a sample containing the macromolecules into one end of the capillary channel and; applying a voltage gradient across the length of the capillary channel, whereby the macromolecules in the sample are separated in the capillary channel. The patent also discusses the use of silica-adsorbing polymers in capillary electrophoresis. U.S. Pat. No. 6,042,710 (Dubrow) discloses a method of manufacturing a microfabricated channel system, the method comprising: providing a device comprising at least one microchannel; and, disposing a polymer in the at least one microchannel, the polymer comprising a net charge of between about 0.01% and 2%, the net charge being of the same charge as at least one surface of the microchannel.
Another means for suppressing undesired analyte-wall interactions invoices the use of coatings to alter the polymer-wall interface. U.S. Pat. No. 5,665,216 (Karger et al.), for example, concerns a coated capillary column containing a UV-transparent polymer network for high performance electrophoretic separation and high sensitivity detection of SDS-proteins comprising: a capillary having an interior cavity and a wall with an inner surface; a layer of coating material on the inner surface of the wall; a UV-transparent hydrophilic polymer network filling the interior cavity; and a UV-transparent buffer, the buffer being selected from a group consisting of Tris-CHES, MES-Na, and AMPD-cacodylic acid (CACO).
Bean, S. R. et al. (1999) (“SODIUM DODECYL SULFATE CAPILLARY ELECTROPHORESIS OF WHEAT PROTEINS. 1. UNCOATED CAPILLARIES,” J. Agric. Food Chem 47(10):4246-55) describes the use of high molecular weight non-cross-linked dextran polymers in a capillary electrophoresis system employing SDS and Tris-borate buffers (pH=8.5). The reference describes the ability of organic additives (e.g., ethylene glycol) to improve the composition's ability to separate proteins. Wu, D. et al. (1992) (“SODIUM DODECYL SULFATE-CAPILLARY GEL ELECTROPHORESIS OF PROTEINS USING NON-CROss-LINKED POLYACRYLAMIDE,” J. Chromatogr. 608:349-356) discusses the use of non-crosslinked polyacrylamide in uncoated capillary electrophoresis systems, buffered with a Tris-borate buffer. Lausch, R. et al. (1993) (“RAPID CAPILLARY GEL ELECTROPHORESIS OF PROTEINS,” J. Chromatogr. 654:190-195) discusses the use of 2×106 MW dextran in capillary gel electrophoresis in a Tris-CHES buffer system. Manabe, T. et al. (1998) (“SIZE SEPARATION OF SODIUM DODECYL SULFATE COMPLEXES OF HUMAN PLASMA PROTEINS BY CAPILLARY ELECTROPHORESIS EMPLOYING LINEAR POLYACRYLAMIDE As A SIEVING POLYMER,” Electrophoresis 19:2308-16) discusses the use of linear polyacrylamide in capillary gel electrophoresis. Ganzler, K. et al. (1992) (“High-Performance Capillary Electrophoresis of SDS-Protein Complexes Using UV-Transparent Polymer Networks,” Anal. Chem. 64:2665-2671) discusses the use of dextran (2×106 MW) and non-crosslinked polyacrylamide in capillary electrophoresis with UV transparent buffers (AMPD-CACO or Tris-CHES).
Unfortunately, two main problems limit the use of CGE. First, only certain polymers are capable of separating polynucleotides and proteins, and many bind only poorly to the capillary surface. Thus, separations are marred by undesired capillary surface electroosmotic flow and surface absorptions. For example, one difficulty encountered in the use of cellulose derivatives in CGE involves the need to suppress capillary surface electroosmotic flow. Prior-employed PEG and dextrans cannot sufficiently suppress capillary surface electroosmotic flow, so acidic pH (pH 2.5) must be applied in order to reduce electroosmotic flow and surface absorptions. Second, glass, commonly used as the capillary material, possess silanol groups that will ionize in water at pH>3. The dissociation of the silanol groups generates a negative charge on the inner surface of the capillary and promotes undesired electroosmotic flow, wall adsorption and peak tailing.
Thus, despite all prior efforts, a need remains to identify polymer compositions that would facilitate improved capillary electrophoretic separations of biomolecules, and in particular, would facilitate high resolution separation for a broad range of proteins and DNA molecules. It would also be desirable that the same separation medium be capable of suppressing electroosmotic flow and reducing analyte-wall interactions, so that no capillary coating would be required. The present invention is directed to these and other goals.